AML1/RUNX1 Point Mutation Possibly Promotes Leukemic Transformation in Myeloproliferative Disorders.

Abstract

Abstract 1902Poster Board I-925Myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages; they are associated with a high frequency of the Janus kinase 2 (JAK2) mutation V617F (JAK2V617F). Some patients with MPD exhibit leukemic transformation (LT) after several years of disease, but the mechanism of LT has been a matter of some controversy due to insufficient insight into the underlying molecular pathogenesis. LT may be a natural sequela of these diseases, whereas treatment with alkylating agents, hydroxycarbamide or their combination may increase the risk of LT. Previous studies have reported that chromosomal abnormalities including -5/5q- and -7/7q- were frequently detected in patients with MPD at the time of LT, suggesting that the development of cytogenetic abnormalities may be associated with the LT of patients with MPD. However, the genes involved in LT still remain obscure. Therefore, we attempted to identify gene alterations involved in LT from patients in the chronic phase (CP) of MPD. Patients with MPD were diagnosed and classified according to the WHO criteria between 1985 and 2007. Patients with PV, ET, PMF, MPD unclassified (MPD-U) and LT of these diseases were examined as approved by the institutional review board at Hiroshima University. Patients gave written informed consent, according to the Declaration of Helsinki. Mutations of JAK2V617F, AML1, CEPBA, FLT3, N-RAS, c-KIT, PTPN11 and TP53 were screened and were identified. Among 417 patients with MPD, 18 (4.3 %) patients progressed to leukemia. At LT, 13 of the 18 patients showed additional cytogenetic abnormalities, including -7/7q- with trisomy 21, inv(3)(q21q26), i(17)(q10) and t(11;19)(q23;p13.3), which are known to be associated with therapy-related leukemia. JAK2V617F was detected in 10 of 14 patients at LT. No patients had a JAK2 mutation pattern that changed during CP to LT. We analyzed gene mutations that may play an important role in leukemogenesis and found five AML1 mutations, one PTPN11 mutation and one CEBPA mutation in patients at the LT, whereas no mutation was detected in patients at CP. The gene alterations were detected at LT in both JAK2V617F-positive and −negative MPD patients, raising the possibility that the hematopoietic stem cells (HSCs) may have been transformed into leukemic blasts as a result of gene abnormalities. Among these gene abnormalities in the MPN patients, we focused on AML1 mutations. To clarify the leukemogenic effect of AML1 mutants, the AML1D171N mutant was transduced into CD34+ cells from eight patients in the CP of MPD using retrovirus transduction methods. The effect of this mutant on cell differentiation/proliferation was assessed by CFC re-plating assays. The D171N plates contained fewer erythroid colonies and more myeloid colonies than the controls. After re-plating, new colonies were detected on all D171N plates but on only few control plates. The D171N-transduced cells contained more CD34+ cells and proliferated more strongly than the controls. These results indicate that the D171N mutant has the potential to increase the myeloid immature cells and to enhance their self-renewal capacity. Furthermore, D171N-transduced cells retained more CD34+ cells than the controls after long-term culture on MS5 stroma cells, and showed significantly more colonies in long-term culture-initiating cells experiments. In this study, AML1 point mutations were detected with high frequency in patients at the LT from both JAK2V617F-positive and -negative MPD. Furthermore, the AML1D171N mutant transduced into CD34+ cells from MPD patients promoted proliferation of primitive progenitors, i.e. leukemic stem cells. These results indicate that AML1 point mutations may have a leukemogenic potential in JAK2V617F-positive stem cells or in pre-JAK2 stem cells, and they may promote leukemic transformation in MPD. Disclosures:No relevant conflicts of interest to declare.